This proposal outlines a research career development plan for a physician-scientist interested in the molecular biology of immunoproteasomes. It relies on a supportive research environment at Cincinnati Children's Hospital Medical Center (CCHMC). The sponsor, Dr. Robert Colbert, has related interests in antigen processing as it applies to the pathogenesis of HLA-B27-associated autoimmune disease. This proposal will provide an expanded knowledge base in molecular immunology via lab meetings, journal clubs, and research seminars, and it includes a new collaboration with Dr. Jeff Molkentin in the Dvision of Molecular Cardiovascular Biology at CCHMC. Immunoproteasomes are specialized for optimal MHC class I antigen processing. By virtue of their role in processing self-antigens, immunoproteasomes are potential targets in the treatment of autoimmune disease. Cooperative assembly of immunoproteasomes ensures their homogeneity in cells that also express constitutive proteasomes that serve many housekeeping functions. Cooperative assembly is dependent on differences between the propeptides of immunosubunits vs. constitutive subunits. We hypothesize that these propeptides mediate their effects through differential propeptide-protein interactions that are the focus of this project. In Aim 1, functionally important sequence differences between homologous propeptides will be identified by testing chimeric propeptides in whole cell assembly assays. In Aim 2, differential interactions between homologous propeptides and an assembly chaperone, proteassemblin, will be characterized using a yeast two-hybrid interaction trap assay. In Aim 3, a novel protein component of early pre-immunoproteasomes will be identified by peptide fingerprinting and characterized by co-immunoprecipitation. Characterizing the mechanisms of cooperative immunoproteasome assembly will serve a long-term goal of identifying targets for manipulating the assembly of immunoproteasomes while leaving vital constitutive proteasome assembly intact.